The long range objective of this research is to understand the mechanisms by which steroid hormones direct the transcription of genes. This proposal is designed to examine the molecular basis for the biological effects of estrogen during avian reproduction. The yolk protein genes are all responsive to estrogen; however, the kinetics of induction vary. Following the administration of estrogen, very low density apolipoprotein II (apo-II) RNA appears within 2-3 hours while there is a lag of about 20 hours before vitellogenin I (VTG-I) RNA can be measured. The basis for these differences is completely unknown. The proposed experiments are designed to examine the role of estrogen response elements, the requirement for additional transcriptional factors, and to determine whether such differences reflect primary and/or secondary effects of estrogen. To accomplish this goal, information about the regulatory regions of several genes is required. The VTG-II gene has been characterized in terms of regulatory components and gene structure. Sequence analysis and footprinting data are available for the apo-II gene, but functional analysis remains to be accomplished. The specific aims of this proposal are to 1) Identify sequences of the apo-II locus that direct transcription of the gene in functional assays, 2) Isolate and characterize genomic clones coding for the VTG-I gene, 3) Identify sequences of the VTG-I locus that direct transcription of the gene in functional assays, and 4) Determine the basis for the slow response to estrogen of the VTG-I gene compared to the rapid response of the apo-II gene. These studies will rely predominately on functional analysis of the DNA sequences by transfection into the estrogen responsive human hepatoma cell line, HepG2. A novel approach will be used in which two different reporter genes will be cotransfected to allow analysis in the same cells. Steroid hormones are crucial in reproduction and development, as well as in health and disease. Basic information concerning the mechanisms of steroid regulation should provide a foundation for understanding the growth of hormone dependent tumors, and may lead to improved drug therapy for estrogen dependent breast tumors.